CN106868020A - Applications of the soybean co-chaperone protein coding gene GmHSP40 on regulation and control flowering of plant - Google Patents

Abstract

The present invention relates to a kind of co-chaperone protein coding gene GmHSP40 from soybean in the control temporal application of flowering of plant.The cDNA nucleotide sequences of GmHSP40 are as shown in SEQ.1；Using recombinant DNA technology construct overexpression GmHSP40 carrier and by transgenic technology obtain either the long-day still under the conditions of short-day florescence significantly postpone transgenic Arabidopsis plants；The present invention also demonstrates that overexpression GmHSP40 is the expression by negative regulatory factor FLC and the FLM encoding gene of induced flowering, and inhibits the expression of the positive regulatory factor FT and SOC1 encoding genes bloomed, postpones so as to cause arabidopsis to be bloomed significantly.GmHSP40 can be used to regulate and control the flowering of plant time and disclose the molecule mechanism of regulation and control flowering of plant.

Description

Soybean co-chaperone protein coding gene GmHSP40 is on regulation and control flowering of plant Application

Technical field

The present invention relates to plant genetic engineering field, main application protection soybean co-chaperone protein coding gene Applications of the GmHSP40 on regulation and control flowering of plant.

Background technology

HSP40 albumen (heat shock protein 40), is found in E.coli, and its molecular weight is about It is 41kDa, it is HSP70 albumen chaperone (Qiu etc., 2006.The diversity of the DnaJ/Hsp40 altogether family,the crucial partners for Hsp70 chaperones[J].Cellular&Molecular Life Sciences Cmls,63(22)2560–2570.).Hsp40 albumen and HSP70 protein-interactings (Hennessy etc., 2005.Rational mutagenesis of a 40kDa heat shock protein from Agrobacterium tumefaciens identifies amino acid residues critical to its in vivo function [J].International Journal of Biochemistry&Cell Biology,37(1):177-191.).Existed later In most of eucaryote, including animals and plants, yeast and the mankind etc., its homologous protein can be found, they all contain one Individual very conservative J domains.Substantial amounts of research shows that J albumen plays important in the everyway such as grow of organism Effect (Orme etc., 2001.A novel plastid-targeted J-domain protein in Arabidopsis thaliana[J].Plant Molecular Biology,46(5):615-626.)。

HSP70 is made up of ATPase active structure domains, the changeable domain of C-terminal and polypeptide binding structural domain, the ATP of HSP70 Folding, the maintenance of protein correct conformation, the transhipment of protein, assembling and dissociation of the enhancing of enzymatic activity to nascent protein were waited Journey is all necessary (Caplan etc., 1993.Eukaryotic homologues of Escherichia coli dnaJ:a diverse protein family that functions with hsp70 stress proteins[J].Molecular Biology of the Cell,4(6):555-563.).And the atpase activity of HSP70 albumen is relatively low, HSP40 albumen can be adjusted The atpase activity of HSP70 is so as to promote HSP70 albumen to complete its various functions, and being structurally characterized in that for this and HSP70 itself is related (Fan etc., 2003.Mechanisms for regulation of Hsp70 function by Hsp40 [J] .Cell Stress&Chaperones,8(4):309-316.).Because ATPase active regions can not only be combined with ATP, while also can be with ADP is combined.So, Hsp70 has ADP-Hsp70 (ADP bonding states, substrate affinity is higher) and ATP-Hsp70, and (ATP is combined State, the substrate affinity of the latter is relatively low) two states (Mayer etc., 2005.Hsp70 chaperones:Cellular functions and molecular mechanism[J].Cellular&Molecular Life Sciences,62:670 ~684.).And HSP40 can result in Hsp70 and change (Laufen etc., 1999.Mechanism of between both the above state regulation of hsp70 chaperones by DnaJ co-chaperones[J].Proceedings of the National Academy of Sciences of the United States of America,96(10):5452- 5457.), when the ATPase active regions of HSP40 and Hsp70 act on, ATPase activity is strengthened promoting ATP to hydrolyze to form ADP, makes ATP-Hsp70 is changed into ADP-Hsp70 forms.This change can influence the change of many binding domain polypeptide conformations of Hsp70, so as to strengthen Its substrate affinity.J domains, as the mark of J albumen, (contain what is extremely guarded by 4 α spirals and 1 HPD module His, Pro, Asp sequence) composition (Rajan etc., 2009.Arabidopsis thaliana J-class heat shock proteins:Cellular stress sensors[J].Functional&Integrative Genomics,9(4):433- 446.).According to protein structure domain, J albumen can be divided into I, II and III 3 hypotype (Walsh etc., 2004.The J-protein family:modulating protein assembly,disassembly and translocation[J].Embo Reports,5(6):567-571.), I type J albumen has 4 kinds of domains：Spiral helicine J domains, G/F (glycine/phenylpropyl alcohol Propylhomoserin) domain, CXXCXGXG Zinc finger domains and carboxyl terminal area；II type J albumen does not have Zinc finger domain；And III type J J domain of the albumen only containing spiral.In addition, IV type J albumen is also found recently, it is referred to as J-like albumen, its structure It is similar to J domains with sequence, but it does not have HPD modules, the HPD module quilts of such as arabidopsis JLP2 (J-like protein2) His, Val, Asp sequence (HVD) replace (Peng etc., 2010.Magmas gene structure and evolution [J].Silico Biology,5(3):251-263.)。

In arabidopsis, find there are 120 HSP40 altogether, these albumen have been reported and have been located at different subcellular fractions Compartment and participate in various bioprocess (Rajan etc., 2009.Arabidopsis thaliana J-class heat shock proteins:Cellular stress sensors[J].Functional&Integrative Genomics,9(4):433- 446.).In plant cell, HSP40 is widely present in the organelles such as mitochondria, chloroplaset, cytoplasm and endoplasmic reticulum.According to Bioinformatic analysis, it is contemplated that have 50 HSP40 albumen to be positioned in cytoplasm, 19 are located in mitochondria, and 12 green in leaf In body, 93 are located on cytoskeleton in endoplasmic reticulum, and 1 is located on plasma membrane, and 24 in nucleus and 2 in liquid (Rajan etc., 2009.Arabidopsis thaliana J-class heat shock proteins in bubble:Cellular stress sensors[J].Functional&Integrative Genomics,9(4):433-446.).But, it is only few The function of number J domain proteins be determined it is reported that plant J domain proteins participate in various stress reactions (Yang etc., 2010.The Arabidopsis chaperone J3regulates the plasma membrane H(+)-ATPase through interaction with the PKS5 kinase[J].Plant Cell,22(4):1313-1332.), and Motion (Noriyuki etc., 2005.An auxilin-like the J-domain protein, JAC1, regulates of chloroplaset phototropin-mediated chloroplast movement in Arabidopsis[J].Plant Physiology, 139(1):151-162.).In host-pathogen interaction process, virus coat and motor protein and J albumen phase interactions With, respectively facilitate virus assembling and motion (Shimizu etc., 2009.Identification of a novel tobacco DnaJ-like protein that interacts with the movement protein of tobacco mosaic virus[J].Archives of Virology,154(6):959-67.)。

Controlling the signal pathway of Arabidopsis plant flowering time mainly has four：Vernalization approach (Vernalization Pathway), Photoperiod pathway (Photoperiodic Pathway), gibberellin pathway (Gibberellin Pathway) with And spontaneous approach (Autonomous Pathway) (Song etc., 2013.Flowering time regulation: photoperiod-and temperature-sensing in leaves[J].Trends Plant Sci.18(10):575- 83).FLOWERING LUCUS (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO I (SOC1) are respectively light week The target gene of CONSTANS (CO) and FLOWER LOCUS (FLC) regulatory factor in phase approach, in each regulatory pathway downstream tool There is Accelerate bloom, be the common regulation and control integration factor that becomes civilized of these approach of blooming, suppressing its expression can cause arabidopsis Postponement is bloomed (Song etc., 2013.Flowering time regulation:photoperiod-and temperature- sensing in leaves[J].Trends Plant Sci.18(10):575-83).FLC is important negative in vernalization approach Regulatory factor, in plant signaling transduction, growing especially regulates and controls the process bloomed and plays an important role, and the promotion with CO is made With just conversely, it is one into flower suppressor, mainly by suppressing the expression of SOC1 and FT and late blooming (Hepworth Deng 2015.Flowering Locus C's Lessons:Conserved Chromatin Switches Underpinning Developmental Timing and Adaptation[J].Plant Physiol.168(4):1237-45)。

The content of the invention

Present invention generally provides a kind of soybean co-chaperone protein coding gene GmHSP40 on regulation and control flowering of plant Application.

The present invention solves the technical scheme that its technical problem is used：

A kind of cDNA nucleotide sequences of GmHSP40 genes such as SEQ ID NO:Shown in 1.

A kind of protein of GmHSP40 gene codes, its amino acid sequence such as SEQ ID NO:Shown in 2.

Compiled by amplifying GmHSP40 total lengths in the total serum IgE that RT-PCR is extracted from soybean Williams82 plant leafs Code sequence (Glyma15g057880.1), the primer is GmHSP40-F, GmHSP40-R.The coded sequence of above-mentioned amplification is first First it is cloned into pENTR/D entry vectors (Invitrogen, http://www.invitrogen.com) in producing pENTR/D/ GmHSP40, is then reacted by LR and recombinates to double base purpose carrier PB7WG2,0 (Karimi etc., 2002 Insert Fragment GATEWAYTM vectors for Agrobacterium-mediated plant transformation.Trends Plant Sci.7,193-195.) middle generation PB7WG2,0-GmHSP40.By binary vector PB7WG2,0-GmHSP40 convert to In Agrobacterium (GV3101) bacterial strain, then by flower-dipping method (Clough, S.J etc., 1998 Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis Thaliana.Plant J.16,735-743.) to go to and obtain GmHSP40-OE transgenic Arabidopsis plants in arabidopsis Col-0. By Phenotypic Observation, it has been found that the transfer-gen plant bolting of overexpression GmHSP40 and bloom than the Col-0 wild types of same age in days Plant significantly postpones, and illustrates that overexpression GmHSP40 inhibits blooming for Arabidopsis plant.Using above-mentioned characteristic, can be by GmHSP40 The cDNA nucleotide sequences of gene or the carrier containing overexpression GmHSP40 full-length cDNAs apply promote or postpone crops and On the flowering time of the ornamental plants such as flowers and in the molecular mechanism of research flowering of plant.

Invention has the advantages that：The flowering of plant time can be regulated and controled simultaneously using the characteristic of encoding gene GmHSP40 Disclose the molecule mechanism of regulation and control flowering of plant.

Brief description of the drawings

Fig. 1 is that the T-DNA regional structures of overexpression soybean GmHSP40 full-length cDNA carriers PB7WG2,0-GmHSP40 are illustrated Figure；

Fig. 2 is that Genomic PCR proves two genes of transgenic arabidopsis strain of GmHSP40-OE1 and GmHSP40-OE2 Soybean GmHSP40 genes are carried in group, arabidopsis Actin genes are compareed as reference gene；

Fig. 3 is two transgenic arabidopsis strains (GmHSP40-OE1 and GmHSP40-OE2) evening of overexpression GmHSP40 Become civilized Phenotypic Observation；

Fig. 4 is the lotus of Col-0 and GmHSP40-OE1 and GmHSP40-OE2 transgenic Arabidopsis plants long-day conditions The comparing of the seat number of sheets and number of days of blooming；

Fig. 5 is the lotus of Col-0 and GmHSP40-OE1 and GmHSP40-OE2 transgenic Arabidopsis plants short-day conditions The comparing of the seat number of sheets and number of days of blooming；

Fig. 6 is that RT-PCR detection arabidopsis is bloomed differential expression of the related gene in Col-0 and GmHSP40-OE；

Fig. 7 is that qRT-PCR detection arabidopsis is bloomed differential expression of the related gene in Col-0 and GmHSP40-OE；

Fig. 8 is the accumulating level of miR156 and miR172 in Col-0 and GmHSP40-OE1 and GmHSP40-OE2 plant Compare.

Specific embodiment

The invention will be further described below in conjunction with the accompanying drawings：

Compiled by amplifying GmHSP40 total lengths in the total serum IgE that RT-PCR is extracted from soybean Williams82 plant leafs Code sequence (Glyma15g057880.1), the primer is GmHSP40-F, 5 '-CACCAAAATGGATGGTCACGGAGGAG- 3′；GmHSP40-R,5′-ATGACCCTTAACCTTATCATCTGCA-3′.The coded sequence of above-mentioned amplification is cloned into first To produce pENTR/D/GmHSP40 in pENTR/D entry vectors (Invitrogen), then reacted the Insert Fragment by LR Restructuring produces PB7WG2,0-GmHSP40, as shown in Figure 1 to double base purpose carrier PB7WG2 in 0.By binary vector PB7WG2, 0-GmHSP40 is converted into Agrobacterium (GV3101) bacterial strain, in then going to arabidopsis Col-0 by flower-dipping method, concrete operations It is as follows：

Agrobacterium-mediated Transformation：Plus 1 μ g plasmids in 100 μ l GV3101 competence；Liquid nitrogen ice swashs 5min, 37 DEG C of water-bath thermal shocks 5min；Plus 800 μ l LB Liquid Cultures be based on step (3) in shake 2-4h in 28 DEG C；Centrifugation 4000rpm, 1min collects thallines；Abandon Except supernatant, 100 μ l LB culture mediums are added gently to suspend；Bacterium solution is coated on flat board, 28 DEG C are cultivated 2-3 days.

The transgenic arabidopsis strain of flower-dipping method initiative overexpression GmHSP40：By transformed Agrobacterium containing anti- Monoclonal is grown on raw element screening and culturing medium, to determining that the successful monoclonal of conversion contains in 5ml liquid YEP mediums after identification (antibiotic) 28 DEG C of activation culture 16h；Take appropriate (1:50) bacterium solution of activation culture (contains antibiosis in 50ml liquid YEP mediums Element) in 28 DEG C of Amplification Culture 6h；By the centrifugation of 200ml agrobatcerium cell 4000rpm, 10min room temperature, 1 times of volume is suspended in 0.05%Silwet L-77,10mM MgCl₂, 5% sucrose solution (i.e. 100ml H₂O adds Silwet L-77 5ml, MgCl₂。 6H₂O 0.202g, sucrose 5g)；Agrobacterium suspension is filled the small lid of 50ml centrifuge tubes, by arabidopsis traverse when infecting , the suspension 10s in making inflorescence invade small lid, weak vibrations tegillum, 200ml agrobacterium suspensions are at least effective for turning Change 300 plants of arabidopsis, it is more than 1500 inflorescences, the step for guarantee to see bacterium solution on plant；Plastic foil bag rolls plant The plant allowed after infecting keeps 16-24h high humility dark 24h；Next day removes the plastics of parcel, and the arabidopsis after infecting is put back to Greenhouse, then collects arabidopsis seed；The seed that vernalization is collected into, plants in pallet, waits after growing green seedlings, sprays Basta herbicides, what can be survived is the arabidopsis strain of required transgenosis.Conversion and base by Agrobacterium Because group PCR checkings (Fig. 2) has obtained two independent transgenic line GmHSP40-OE1 and GmHSP40-OE2.

Good Col-0, GmHSP40-OE1 and GmHSP40-OE2 seed of vernalization is uniformly layered on into 1/2MS+1.5%Agar to put down On plate, title and the date of good each material are marked respectively, sealed flat board with sealed membrane, be disposed vertically in arabidopsis artificial lighting Incubator (illumination condition：22 DEG C, 16h；Dark condition：20 DEG C, 8h；Humidity 85%) on culture dish frame, after it is cultivated one week, Seedling is transplanted in soil respectively.Long-day：To transplant to Col-0, GmHSP40-OE1 and GmHSP40-OE2 seedling in soil (cave seed plate) is placed in arabidopsis artificial lighting incubator, and its condition of culture is：Illumination condition：22 DEG C, 16h；Dark condition：20 DEG C, 8h；Humidity 85%.When Arabidopsis plant firm bolting 1-2cm, the number of days and corresponding of blooming of each plant is counted respectively The lotus throne number of blade, such as Fig. 4；Short-day：To transplant to Col-0, GmHSP40-OE1 and GmHSP40-OE2 seedling (the cave seedling in soil Disk) arabidopsis artificial lighting incubator is placed in, its condition of culture is：Illumination condition：22 DEG C, 8h；Dark condition：20 DEG C, 16h； Humidity 85%.When Arabidopsis plant firm bolting 1-2cm, bloom number of days and the corresponding lotus throne leaf of each plant are counted respectively Piece number, such as Fig. 5.

Research shows that the number of blade is that this is due to once for distinguishing the photoperiod to flower development and the effect of flower induction Startup is bloomed, and lotus throne leaf (Chen Xiao etc., molecule during 2006. photoperiods influence plant flowers will not be again grown on arabidopsis stem Mechanism [J] northwests Botany Gazette, 26 (7):1490-1499.).By Phenotypic Observation, it has been found that overexpression GmHSP40's turns Gene plant bolting and bloom than the notable delay of the Col-0 WT lines (shown in Fig. 3) of same age in days.Our statistical comparisons two The lotus throne number of sheets of person and number of days of blooming, as a result show, either under the conditions of long-day or short-day, GmHSP40-OE turns base Because the lotus throne of Arabidopsis plant significantly increases than the lotus throne number of blade of Col-0 WT lines；And under long-day conditions The number of days of blooming of GmHSP40 transgenic Arabidopsis plants delays at least 15 days than Col-0, under the conditions of short-day The number of days of blooming of GmHSP40-OE transgenic Arabidopsis plants delays at least 30 days than Col-0.Illustrate overexpression GmHSP40 Inhibit blooming for Arabidopsis plant, overexpression soybean GmHSP40 causes Arabidopsis plant to be bloomed significantly to postpone GmHSP40-OE The late flowering phenotype of transgenic Arabidopsis plants.

Genomic DNA is extracted using CTAB methods：1g Arabidopsis leafs are taken, is moved into 1.5mLEppendorf pipes, use mortar Grind sample and cause pulpous state；400-500 microlitres of CTAB Extraction buffers are added, is mixed (CTAB is preheated in 65 DEG C of water-baths), every 5 points Clock gently shakes several times, 12000r/min centrifugations 10min after 20min；Careful Aspirate supernatant, adds isometric chloroform (each 400 microlitres) solution, mix (using concussion instrument), 4 DEG C, 12000r/min, 10min is centrifuged；The bodies such as careful Aspirate supernatant, addition Long-pending isopropanol, is mixed, 4 DEG C, 12000r/min with concussion instrument, and 10min is centrifuged；Abandoning supernatant, washs heavy with 70% ethanol Form sediment once；(5-15min is typically dried after drying at room temperature) to be dissolved in 30-50 microlitres of deionized water, mix, centrifugation, in -20 DEG C Or saved backup at -70 DEG C.Two transgenic arabidopsis strains of GmHSP40-OE1 and GmHSP40-OE2 are proved by Genomic PCR Soybean GmHSP40 genes are carried in the genome of system, as shown in Figure 2.

For the differential expression of related gene of being bloomed in studying Col-0 plant and GmHSP40-OE transfer-gen plants, we Bloomed between have detected Col-0 and GmHSP40-OE transfer-gen plants by RT-PCR and qRT-PCR the differential expression of related gene. Specific method is as follows：

First, arabidopsis RNA is slightly carried using organic reagent Trizol：1g Arabidopsis leafs are taken to be put into mortar (mortar is used Before should first clean and dry, add a small amount of absolute ethyl alcohol and light it is carried out disinfection, place into afterwards cold in advance in liquid nitrogen Freeze) in, add appropriate liquid nitrogen (all of blade should be submerged) in vaned mortar to filling, blade is pressed into a side in liquid nitrogen To quickly powder is ground to, powder is then rapidly transferred in the centrifuge tube of 1.5mL (carrying RNA special) (centrifuge tube exists Preferably first soaked with liquid nitrogen using preceding, and the powder in pipe is no more than 0.5mL as far as possible)；Ventilating kitchen is opened, in ventilating kitchen In state in centrifuge tube each Trizol reagents (Trizol has severe toxicity, be should be specifically noted that during dosing product) for adding 1mL rapidly upward, stand It is placed on turbula shaker and strongly vibrates, it is fully mixed, often vibrate and just open for a moment centrifugation lid deflation, prevents centrifugation Pipe can expand with heat and contract with cold；Centrifuge is cooled to 4 DEG C in advance in advance, centrifuge tube is put into centrifuge after vibration, in 4 DEG C of 12000r/min Under the conditions of 10min is centrifuged.During supernatant in pipe after centrifugation moved into a new 1.5mL centrifuge tube, place at room temperature 5min；To 200uL chloroforms (chloroform) is added in the above-mentioned new centrifuge tube equipped with supernatant, lid, vortex oscillation instrument are covered tightly Upper strength vibrates 15s, after it is sufficiently mixed, 2-3min is stood at room temperature, solution layering is occurred；Then at 4 DEG C 15min is centrifuged under the conditions of 12000r/min.After centrifugation, occur layering (liquid phase and organic phase) in centrifuge tube, taking-up gently from Heart pipe, should not allow the part of layering to mix up, and centrifuge tube is inclined into 45 °, the superiors' liquid is gently suctioned out with liquid-transfering gun and is put into one In new 1.5mL centrifuge tubes, note necessarily being drawn onto intermediate layer and lower floor's liquid；Added and supernatant toward above-mentioned centrifuge tube The aqueous isopropanol of same volume, places 10min at room temperature, settles RNA, then under the conditions of 4 DEG C of 12000r/min from Heart 10min；Supernatant (careful to draw, precipitation should not to be directed at and drawn) is removed after centrifugation, retains precipitation.If can not thoroughly draw Clearly, 2min can somewhat be centrifuged again with centrifuge, continues to draw supernatant；The another centrifuge tube for taking a new 1.5mL, is separately added into The alcohol and 250ul RNase-free water of 750ul100%, fully piping and druming are mixed；To equipped with precipitation centrifuge tube in plus Enter 1mL is prepared 75% ethanol, gently blown and beaten with liquid-transfering gun, precipitation is suspended, then under the conditions of 4 DEG C of 7500r/min Centrifugation 5min；Removal supernatant, stays precipitation, room temperature to place 2-3min.Again to addition 30uL RNase-free water in pipe with molten Solution RNA, is gently blown and beaten with pipette tips, makes its uniform dissolution.

By the step of being given on extracts kit (RNeasy Plant Mini Kit.QIAGEN) specification of RNA pairs RNA is purified.The preparation done is needed before experiment：β-ME (beta -mercaptoethanol) Buffer RLT should be added before, 10uL β-ME (1mL RLT/10uL β-ME) should be added in per 1mL Buffer RLT；When first switching on Buffer RPE, should Pointed out according to label above, inwardly add 4 times of ethanol (100%) of volume (44mL), rocked up and down well mixed；Prepare DNA digestive ferment DNase I Stock Solution remove the water (RNase-free of RNase to addition 550uL in DNase I bottles Water), gently rock 3-5 times back and forth, mix, powder is substantially dissolved in RNase-free water, then take 70uL Buffer RDD and 10uL DNase I Stock Solution piping and druming is placed on ice after mixing；In preservation in -20 DEG C of refrigerators DNase I Stock Solution.Extract specific steps：(1) 70uL RNase-free water are added to and above-mentioned are slightly carried The 30uL RNA for getting, then the Buffer RLT of 350uL are added thereto to, it is well mixed it；(2) in centrifuge tube in (1) The 100% of 250uL ethanol solution is added, is gently blown and beaten with pipette tips after it is mixed completely, rapidly turn the interior 700uL solution of pipe Move on in the RNeasy spin column of 2ml pink colours, after closeing the lid, 15s be centrifuged under the conditions of 4 DEG C of 12000rpm, treat from After the heart, liquid in pipe is discarded；(3) 350uL Buffer RW1 solution is added in above-mentioned pink colour RNeasy spin column, 15s is centrifuged under the conditions of 4 DEG C of 12000rpm, after being centrifuged, liquid in pipe is discarded；(4) it is new take a 1.5mL go RNase from Heart pipe, the Buffer RDD of 70uL are added to it, add the DNase I Stock Solution of 10uL, make its mixing equal It is even；(5) the 80uL DNase I Stock Solution mixed liquors that (4) mix are transferred to the RNeasy of pink colour in (3) In spin column, room temperature places 15min；(6) 350uL Buffer RW1 are added to the RNeasy spin of pink colour in (5) In column, 15s is centrifuged under the conditions of 4 DEG C of 12000rpm, after being centrifuged, discards liquid in pipe；(7) to pink colour in (6) RNeasy spin column add 500uL Buffer RPE, and 15s is centrifuged under the conditions of 4 DEG C of 12000rpm, after being centrifuged, abandon Fall liquid in pipe；(8) the Buffer RPE solution of 500uL is continuously added in the RNeasy spin column of pink colour in (7), 2min is centrifuged under the conditions of 4 DEG C of 12000rpm, after being centrifuged, liquid in pipe is discarded；(9) take a 1.5ml RNase-free from Heart pipe, lid is cut, and is then placed on it the RNeasy spin column of pink colour in (8), and to adding 30- in pillar The RNase-free water of 50uL, 1min is centrifuged under the conditions of 4 DEG C of 12000rpm, after being centrifuged, discards liquid in pipe；(10) The liquid portion at collecting pipe bottom, the RNA for as purifying, careful absorption is placed in the centrifuge tube of the new RNase-free of 1.5ml.Take 1uL solution determines its RNA concentration and records using NANoDROP 2000C, be stored in -80 DEG C it is standby.

The synthesis of cDNA：Before reverse transcription, RNA degenerative treatments should be first carried out：Taken out from -80 DEG C of refrigerators and be placed in ice with RNA In box, appropriate RNA (rna content is with reference to system in table 1 below) is suctioned out in 200uL centrifuge tubes with liquid-transfering gun pipette tips, PCR is set The condition of instrument is 65 DEG C, 5min, then centrifuge tube is put into PCR instrument, carries out hot change treatment；After thermal denaturation treatment, from PCR instrument Middle taking-up centrifuge tube, and be immediately placed in ice (prevent its renaturation), with reference to the reaction system in table 1 below, added in centrifuge tube Each composition of respective amount；Reverse transcription reaction：The condition for setting PCR instrument is 37 DEG C, 15min；98 DEG C, 5min；Carry out reverse transcription Treatment.After reaction terminates, gained cDNA is put into -20 DEG C of refrigerators and is preserved.

The cDNA synthetic systems of table 1

PCR is expanded：PCR programs are set to " 95 DEG C of 3min of predegeneration；95 DEG C of 30s of denaturation；55 DEG C of 30s of annealing；Extend 72 DEG C 1min；Fully extend 72 DEG C of 5min；Preserve 4 DEG C of 24h ", circulate 34 times.Annealing temperature and extension of time according to each primer of table 2 come Regulation；According to PCR reaction systems (table 3) in 2xTaqPCR Master Mix specifications, reaction solution is prepared；Add corresponding primer And above system enters performing PCR amplification；Glue, electrophoresis.

The primer sequence of table 2

The pcr amplification reaction system of table 3

Quantitative fluorescent PCR is analyzed：With reference to reactant in the specification of THUNDERIRD qPCR Mix kits (TOYOBO) System's (table 4), prepares reaction solution；Quantitative fluorescent PCR parameter is set to " 95 DEG C of 5min of predegeneration；95 DEG C of 5s of denaturation；Extend 60 DEG C 35 circulations of 45s "；Adding primer and above system carries out quantitative fluorescent PCR reaction.

The quantitative fluorescent PCR reaction system of table 4

RT-PCR results show, compared with Col-0 plant, in GmHSP40-OE transfer-gen plants, regulate and control two for blooming The expression quantity of individual main positive regulatory factor encoding gene FT and SOC1 is lowered；And negative regulator gene FLC and FLM expression quantity of blooming exists Substantially (Fig. 6) is raised in GmHSP40-OE transfer-gen plants.Consistent with RT-PCR results, qRT-PCR results are also indicated that In GmHSP40-OE transfer-gen plants, the expression quantity of FT and SOC1 is significantly lowered, and FLM and FLC expression quantity significantly raises (figure 7).These results show expression of the overexpression soybean GmHSP40 induction of the negative regulator gene of correlation of blooming, and inhibit and bloom The expression of related positive regulating gene, causes arabidopsis to be postponed and blooms.

The albumen of FLOWERING LOCUS T (FT) gene code is a kind of plain signaling molecule of blooming of long range operating, Played an important role on starting into flower；FT can directly facilitate arabidopsis and bloom, and be one of the key gene of regulation and control of blooming (Noriko etc., 2011.Expression of FLOWERING LOCUS T from Arabidopsis thaliana induces precocious flowering in soybean irrespective of maturity group and stem growth habit[J].Planta,233(3):561-568.), in arabidopsis, FT overexpression can show plant Go out early blossoming phenotype, and there are evening flower phenotype (Kardailsky etc., 2000.Activation tagging of in its mutant ft the floral inducer FT[J].Science,286(5446):1962-1965.).FT and SOC1 is used as blooming approach most The crucial integration factor in downstream, their expression quantity is significantly reduced；In approach of blooming, FT and SOC1 is to directly facilitate plant The gene bloomed.Overexpression GmHSP40 significantly suppress FT and SOC1 and express, and cause GmHSP40-OE plant late bloomings；And FLC and FLM further suppresses respectively as the most direct negative regulator gene in FT and SOC1 upstreams, the rising of their expression quantity The expression of FT and SOC1, so this is also the reason for blooming most direct in its evening；These results show overexpression soybean GmHSP40 Induction of the expression of the negative regulatory factor encoding gene bloomed, and the expression of the positive regulatory factor encoding gene bloomed is inhibited, Significantly postpone so as to cause to bloom.Overexpression GmHSP40 is direct effect or the table by inducing FLC to the suppression that FT is expressed Up to and realize indirectly need further research.

It is another there are some researches show miR156 has played important function to the nutrient growth for maintaining plant, it and target gene SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) has collectively constituted a new way (Fu for controlling to bloom Deng 2012.Overexpression of miR156 in switchgrass (Panicum virgatum L.) results in various morphological alterations and leads to improved biomass production [J].Plant Biotechnology Journal,10(4):443-452.), SPL can promote the expression of FT and SOC1, and mistake Expression miR156 can suppress the expression of related SPL genes and then suppress the expression of FT and SOC1, postpone plant blossom (Spanudakis etc., 2014.The role of microRNAs in the control of flowering time [J] .Journal of Experimental Botany,65(2):365-380.)；And miR172 has rush to flowering of plant process Enter effect, it can suppress the expression of its target gene TARGET OF EAT (TOE) to control flowering time；In arabidopsis, TOE Overexpression can suppress the expression of FT and SOC1 and plant evening is bloomed (Glazinska etc., 2009.The putative miR172 target gene In APETALA2-like is involved in the photoperiodic flower induction of Ipomoea nil[J].Plant Physiol,166(16):1801-1813.).And our experiment card Accumulation of the accumulating level of real miR156 and miR172 in Col-0 and GmHSP40-OE plant is without significant difference (Fig. 8), explanation It not is to be caused by the Accumulation discrepancy of the two miRNA that GmHSP40-OE plant evenings bloom, but caused by other mechanism.

In addition to the implementation, the present invention can also have other embodiment.All use equivalents or equivalent transformation shape Into technical scheme, all fall within the protection domain of application claims.

<110>Zhejiang Normal University

<120>Applications of the soybean co-chaperone protein coding gene GmHSP40 on regulation and control flowering of plant

<160>4

<210>1

<211>1494

<212>DNA

<400>1

atggatggtc acggaggagg aggaggaggg agcagaggag aagcggagct ctggctttac 60

acagcgaaca aggttttaag cgcacgtgac ctgcacgggg cccgctcatt tgcgatccgg 120

gctcgagact ccgacccgag atacgagccc actgagctcc ttttagcggt gattgatacc 180

ctcatggccg gcgaggcccg gatcaacgac caactggact ggtacgctat tttgcaagtc 240

ctccgttaca ctcagaacat cgactacatc gccgcccagt accgccgcct cgccacccaa 300

ctcgaccctc accacaaccc cttcgccttc gccgctcatg ccttcacgct cgtccacgac 360

gcgtggaccg tactctccaa cccaactaaa aagactttct acgacaacca actccggctc 420

ctcactcaac cccctcctcc tcagccacca ccaccacctc ccgctcctcc tgccccggtg 480

gccttctttc ctattcagcc gccgcaaccg aacctaaacc ccaacccgat tccgaaccta 540

gtccctccaa gggaaagccc taggcctagg cctagggtag aggtggagcc gccaccaccg 600

gcaccgccgc catcgagtca gctggacaat gcgaccgaat tgactcgggc gagtgaggcc 660

gagagcgaag gggcgagttt ctggaccgcg tgcccttact gttacgttat gtacgagtat 720

ccgaaggtgt acgaggattg tactttgcgg tgccagaatt gtcggagggg gtttcatgcg 780

atggttgtac gttctccgcc aaaagatggt acttttggct cgttttgcag ttggggtttt 840

ttccctgtgg gtttttctgg ggattttaag gacattaacg ggtcttcttc caagtggaac 900

cctttctccc ctttgtttcc ttgcgccttg aagggggccg agcagaatag taggtaccag 960

aaggggcctt gggtttttta cgatgatgac gcgtctgcgg agttcgtcga ggctggttcc 1020

gacacgacgg aagatgattc cgatgatgat gactggcgtg gcgggaatca gaaggggacc 1080

acgaggagga ggaaaagaag gaagaggaga agcaatgctg gtggtgatgt tagaagggta 1140

cctacaattg agaggcctag aagacgggtt cagaatagtg acggtaatga cagtgtgggg 1200

aatggtgagg ctgtggatgg tggtgccctt gccgtgccgg tggcaccgga gtctagcaag 1260

aaggctgtgg cgttgggtgg atcaaggagg aggagtgaga ggaacttggg gaagttggat 1320

ttaaatgttg agtttagcaa tgaggtggag gagcctgtgc atggagcagg tgaagggaat 1380

gggaatgctg aggataatat tgaagggatt gggttttttg aggggctgga tgagttcctt 1440

agtagcttgc ctattctcaa tgttgttgca gatgataagg ttaagggtca ttag 1494

<210>2

<211>497

<212>PRT

<213>Artificial sequence

<400>2

Met Asp Gly His Gly Gly Gly Gly Gly Gly Ser Arg Gly Glu Ala Glu

1 5 10 15

Leu Trp Leu Tyr Thr Ala Asn Lys Val Leu Ser Ala Arg Asp Leu His

20 25 30

Gly Ala Arg Ser Phe Ala Ile Arg Ala Arg Asp Ser Asp Pro Arg Tyr

35 40 45

Glu Pro Thr Glu Leu Leu Leu Ala Val Ile Asp Thr Leu Met Ala Gly

50 55 60

Glu Ala Arg Ile Asn Asp Gln Leu Asp Trp Tyr Ala Ile Leu Gln Val

65 70 75 80

Leu Arg Tyr Thr Gln Asn Ile Asp Tyr Ile Ala Ala Gln Tyr Arg Arg

85 90 95

Leu Ala Thr Gln Leu Asp Pro His His Asn Pro Phe Ala Phe Ala Ala

100 105 110

His Ala Phe Thr Leu Val His Asp Ala Trp Thr Val Leu Ser Asn Pro

115 120 125

Thr Lys Lys Thr Phe Tyr Asp Asn Gln Leu Arg Leu Leu Thr Gln Pro

130 135 140

Pro Pro Pro Gln Pro Pro Pro Pro Pro Pro Ala Pro Pro Ala Pro Val

145 150 155 160

Ala Phe Phe Pro Ile Gln Pro Pro Gln Pro Asn Leu Asn Pro Asn Pro

165 170 175

Ile Pro Asn Leu Val Pro Pro Arg Glu Ser Pro Arg Pro Arg Pro Arg

180 185 190

Val Glu Val Glu Pro Pro Pro Pro Ala Pro Pro Pro Ser Ser Gln Leu

195 200 205

Asp Asn Ala Thr Glu Leu Thr Arg Ala Ser Glu Ala Glu Ser Glu Gly

210 215 220

Ala Ser Phe Trp Thr Ala Cys Pro Tyr Cys Tyr Val Met Tyr Glu Tyr

225 230 235 240

Pro Lys Val Tyr Glu Asp Cys Thr Leu Arg Cys Gln Asn Cys Arg Arg

245 250 255

Gly Phe His Ala Met Val Val Arg Ser Pro Pro Lys Asp Gly Thr Phe

260 265 270

Gly Ser Phe Cys Ser Trp Gly Phe Phe Pro Val Gly Phe Ser Gly Asp

275 280 285

Phe Lys Asp Ile Asn Gly Ser Ser Ser Lys Trp Asn Pro Phe Ser Pro

290 295 300

Leu Phe Pro Cys Ala Leu Lys Gly Ala Glu Gln Asn Ser Arg Tyr Gln

305 310 315 320

Lys Gly Pro Trp Val Phe Tyr Asp Asp Asp Ala Ser Ala Glu Phe Val

325 330 335

Glu Ala Gly Ser Asp Thr Thr Glu Asp Asp Ser Asp Asp Asp Asp Trp

340 345 350

Arg Gly Gly Asn Gln Lys Gly Thr Thr Arg Arg Arg Lys Arg Arg Lys

355 360 365

Arg Arg Ser Asn Ala Gly Gly Asp Val Arg Arg Val Pro Thr Ile Glu

370 375 380

Arg Pro Arg Arg Arg Val Gln Asn Ser Asp Gly Asn Asp Ser Val Gly

385 390 395 400

Asn Gly Glu Ala Val Asp Gly Gly Ala Leu Ala Val Pro Val Ala Pro

405 410 415

Glu Ser Ser Lys Lys Ala Val Ala Leu Gly Gly Ser Arg Arg Arg Ser

420 425 430

Glu Arg Asn Leu Gly Lys Leu Asp Leu Asn Val Glu Phe Ser Asn Glu

435 440 445

Val Glu Glu Pro Val His Gly Ala Gly Glu Gly Asn Gly Asn Ala Glu

450 455 460

Asp Asn Ile Glu Gly Ile Gly Phe Phe Glu Gly Leu Asp Glu Phe Leu

465 470 475 480

Ser Ser Leu Pro Ile Leu Asn Val Val Ala Asp Asp Lys Val Lys Gly

485 490 495

His

497

<210>3

<211>26

<212>DNA

<213>Artificial sequence

<400>3

caccaaaatg gatggtcacg gaggag 26

<210>4

<211>25

<212>DNA

<213>Artificial sequence

<400>4

atgaccctta accttatcat ctgca 25

Claims (5)

1. a kind of cDNA nucleotide sequences of GmHSP40 genes such as SEQ ID NO:Shown in 1.

2. a kind of protein of GmHSP40 gene codes, its amino acid sequence such as SEQ ID NO:Shown in 2.

3. a kind of carrier of the overexpression GmHSP40 full-length cDNAs containing described in claim 1, it is characterized by：With pB7WG2,0 It is skeleton carrier.

4. the nucleotide sequence of the GmHSP40 genes according to claim 1,2 or 3, amino acid sequence or carrier are promoting Or the application for postponing on the flowering time of ornamental plant such as crops and flowers.

5. the nucleotide sequence of the GmHSP40 genes according to claim 1,2 or 3, amino acid sequence or carrier are in research Application in the molecular mechanism of flowering of plant.